The relative levels of RANKL and its negative regulator, OPG, are critical for the initiation and progression of osteoclastogenesis. However, factors that modulate the amplitude and tempo of osteoclastogenesis are less well understood, and growing evidence indicates that microRNAs (miRNAs, miRs) play an important role in this process. One miRNA can regulate families of structural or signaling molecules within a particular pathway; thus miRNAs can amplify or dampen the effects of extracellular signals, balancing and buffering cellular responses, in addition to regulating the cross talk between signaling pathways. In the osteoclast lineage, miR-29 family members are highly expressed, are increased during osteoclast differentiation, and promote osteoclastogenesis. Our in vitro data demonstrate that miR-29 promotes osteoclastogenesis, at least in part, by promoting commitment to the osteoclast fate and by supporting cell migration. To address possible mechanisms, we identified a novel set of miR-29 targets with the potential to regulate commitment, cytoskeletal organization, cell motility and osteoclast function. In addition, we developed an in vivo model (miR-29 competitive inhibitor or sponge mice) for studying miR-29 actions in osteoclasts, and these mice display increased trabecular bone volume. We hypothesize that miR-29 promotes osteoclastogenesis, at least in part, by supporting cell migration and lineage commitment, and therefore is essential for normal bone remodeling. In Aim 1, we will perform a comprehensive analysis of miR-29 function during osteoclastogenesis using TRAP-miR-29 sponge mice. We will characterize the skeletal phenotype of miR-29 sponge mice, and determine effects of the miR-29 sponge on lineage commitment, differentiation, apoptosis and resorption in primary cells. Live cell imaging will be used to evaluate parameters of cell motility and fusion. In Aim 2, we will determine the mechanisms by which miR-29 and its targets control osteoclastogenesis, by studying the function of 2 newly validated miR-29 targets that are strongly regulated by RANKL, but have not been previously studied in the osteoclast lineage: SRGAP2 and CD93. Knock down and over expression studies will be performed in vitro and in vivo, and effects on commitment, motility, cytoskeletal organization and resorption will be quantified. Impact: Understanding how miR-29 and its targets regulate osteoclastogenesis will provide important new information about the process of osteoclastogenesis itself. Further, miRNA-based therapeutics represent powerful tools treating disease, and are in clinical trials. A serious limit to their utility is the gap in our knowledge o miRNA targets and regulated networks in multiple cell types. Such information is critical for the development of novel therapeutics, their translation to the clinic, and for predicting efficacy and safety. Some mechanisms that we study in osteoclasts could be active in other cell systems; therefore this work could also contribute to our understanding of the role of the miR-29 family in cancer, aging and diabetes.